1. Field of the Invention
The present invention relates generally to the NOR gene. More specifically, it relates to methods and compositions for the modification of plant phenotypes with the NOR gene.
2. Description of the Related Art
The ripe phenotype is the summation of biochemical and physiological changes occurring at the terminal stage of fruit development rendering the organ edible and desirable to seed dispersing animals and valuable as an agricultural commodity. These changes, although variable among species, generally include modification of cell wall ultrastructure and texture, conversion of starch to sugars, increased susceptibility to post-harvest pathogens, alterations in pigment biosynthesis/accumulation, and heightened levels of flavor and aromatic volatiles (Rhodes, 1980; Hobson and Grierson, 1993). Several of theses ripening attributes translate to decreased shelf-life and high input harvest, shipping and storage practices, particularly via changes in firmness and the overall decrease in resistance to microbial infection of ripe fruit. Currently acceptable techniques for minimizing the consequences of undesirable ripening characteristics include premature harvest, controlled atmosphere storage, pesticide application, and chemically induced ripening to synchronize the timing of maturation. Unfortunately, added production, shipping and processing expenses, in addition to reduced fruit quality, are often the consequence of these practices, challenging both the competitiveness and long term sustainability of current levels of crop production.
Although most fruit display modifications in color, texture, flavor, and pathogen susceptibility during maturation, two major classifications of ripening fruit, climacteric and non-climacteric, have been utilized to distinguish fruit on the basis of respiration and ethylene biosynthesis rates. Climacteric fruit such as tomato, cucurbits, avocado, banana, peaches, plums, and apples, are distinguished from non-climacteric fruits such as strawberry, grape and citrus, by their increased respiration and ethylene biosynthesis rates during ripening (Grierson, 1986). Ethylene has been shown to be necessary for the coordination and completion of ripening in climacteric fruit via analysis of inhibitors of ethylene biosynthesis and perception (Yang, 1985; Tucker and Brady, 1987), in transgenic plants blocked in ethylene biosynthesis (Klee et al., 1991; Oeller et al., 1991; Picton et al., 1993 a), and through examination of the Never-ripe (Nr) ethylene perception mutant of tomato (Lanahan et al., 1994).
Considerable attention has been directed toward elucidating the molecular basis of ripening in the model system of tomato during recent years (reviewed in Spiers and Brady, 1991; Gray et al, 1992 and 1994; Giovannoni, 1993; Theologis 1992 and Theologis et al., 1993). The critical role of ethylene in coordinating cliimactic ripening at the molecular level was first observed via analysis of ethylene inducible ripening-related gene expression (Tucker and Laties, 1984; Lincoln et al., 1987; Maunders et al., 1987; DellaPenna et al., 1989; Starrett and Laties; 1993). Several ripening genes, including ACC synthase and ACC oxidase, have been shown via antisense gene repression to have profound influences on the onset and degree of ripening (Hamilton et al., 1990; Oeller et al., 1991). Although the sum effect of this research has been a wealth of information pertaining to the regulation of ethylene biosynthesis and its role in ripening, the molecular basis of developmental cues which initiate ripening-related ethylene biosynthesis, and additional aspects of ripening not directly influenced by ethylene, remain largely unknown (Theologis et al., 1993).
Single locus mutations which attenuate or arrest the nornal ripening process, and do not ripen in response to exogenous ethylene, have been identified in tomato and are likely to represent lesions in regulatory components necessary for initiation of the ripening cascade, including ethylene biosynthesis (Tigchelaar et al., 1978; Grierson, 1987; Giovannoni, 1993; Hobson and Grierson, 1993; Gray et al., 1994). One such mutation, Nr mutation, has been identified and represents a gene responsible for ethylene perception and/or signal trnasduction and is a tomato homologue of the Arabidosis Ethylene response 1 (Etr1) gene (Yen et al., 1995; Wilkinson et al., 1995).
Tomato has served as a model for ripening of climacteric fruit. Ripening-related genes have been isolated via differential gene expression patterns (Slater et al., 1985, Lincoln et al., 1987, Pear et al., 1989, Picton et al., 1993b) and biochemical function (DellaPenna et al., 1986; Sheehy et al., 1987; ray et al., 1988; Biggs and Handa, 1989; Harriman and Handa, 1991; Oeller et al., 1991; Yelle et al., 1991). Promoter analysis of ripening genes has been performed via examination of promoter/reporter construct activities in transient assay systems and transgenic plants. The result has been the identification of cis-acting promoter elements which are responsible for both ethylene and non-ethylene regulated aspects of ripening (Deikman et al., 1992; Montgomery et al., 1993). Trans-acting factors which interact with these promoters also have been identified via gel-shift and footprint experiments, although none have been isolated or cloned (Deikman and Fischer, 1988; Cordes et al., 1989; Montgomery et al., 1993).
The in vivo functions of several ripening-related genes including polygalacturonase, pectinmethylesterase, ACC synthase, ACC oxidase, and phytoene synthase have been tested via antisense gene repression and/or mutant complementation in transgenic tomatoes. For example, the cell wall pectinase, polygalacturonase, was shown to be necessary for ripening-related pectin depolymerization and pathogen susceptibility, however, the inhibition of PG expression had minimal effects on fruit softening (Smith et al., 1988, Giovannoni et al., 1989, Kramer et al., 990). Significant reduction in rates of ethylene evolution resulting in inhibition of most ripening characteristics was observed in both ACC synthase and ACC oxidase antisense mutants (Oeller et al., 1991; Hamilton et al., 1990). Non-ripening antisense fruit were subsequently restored to normal ripening phenotype with the application of exogenous ethylene.
Further analysis of transgenic tomatoes inhibited in ethylene biosynthesis demonstrates that climacteric ripening represents a combination of both ethylene mediated and developmental control (Theologis et al., 1993). Although antisense ACC synthase tomatoes which failed to produce ethylene did not ripen, gene expression analysis demonstrated that several ripening-related genes, including polygalacturonase and E8 are expressed in the absence of ethylene. This observation confirms the presence of a developmental (or non-ethylene regulated) component of ripening. In fact, an ethylene requirement was observed for translation but not transcription of polygalacturonase mRNA, suggesting interaction between ethylene and non-ethylene components of ripening for expression of at least a subset of ripening genes (Theologis et al., 1993).
While the above studies have provided some insight into the ripening process in plants, there is still a great need in the art for novel methods and compositions for the creation of plants having enhanced phenotypes. In particular, there is a need in the art for the isolation the RIN and NOR genes. The isolation of these genes would allow the creation of novel transgenic plants altered in their fruit characteristics and/or ethylene responsiveness, and having one or more added beneficial properties.
In one aspect, the current invention provides an isolated nucleic acid sequence comprising the NOR gene. In one embodiment of the invention, the NOR gene may be further defined as isolatable from the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO:6 or SEQ ID NO:7. In particular embodiments of the invention, the invention provides an isolated nucleic acid corresponding to an open reading frame of the NOR cDNA, for example, which may be denoted by the nucleotides as indicated by bold letters in FIG. 6.
In another aspect, the invention provides an isolated nucleic acid sequence having from about 17 to about 1209, about 25 to about 1209, about 30 to about 1209, about 40 to about 1209, about 60 to about 1209, about 100 to about 1209, about 200 to about 1209, about 400 to about 1209, about 600 to about 1209, about 800 to about 1209, or about 1000 to about 1209 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:7. Similarly, the invention provides such nucleic acid segments from SEQ ED NO:1. In particular embodiments of the invention, the nucleic acid sequences of SEQ ID NO:6 and SEQ ID NO:7 are provided. In particular embodiments of the invention, a nucleic acid sequence of the invention may further comprising an enhancer, such as an intron. A nucleic acid sequence of the invention may also include a transcriptional terminator. Such sequences may be native to the NOR gene or heterologous from potentially any species.
In yet another aspect, the invention provides an expression vector comprising a NOR gene. Such a NOR gene may be in accordance with any of the NOR-containing sequences described herein. The expression vector may comprise the NOR gene operably linked to a native or heterologous promoter, either in sense or antisense orientation relative to the promoter. Potentially any heterologous promoter may be used, for example, a promoter is selected from the group consisting of CaMV 35S, CaMV 19S, nos, Adh, actin, histone, ribulose bisphosphate carboxylase, R-allele, root cell promoter, xcex1-tubulin, ABA-inducible promoter, turgor-inducible promoter, rbcS, corn sucrose synthetase 1, corn alcohol dehydrogenase 1, corn light harvesting complex, corn heat shock protein, pea small subunit RuBP carboxylase, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, bean glycine rich protein 1, CaMV 35s transcript, Potato patatin, actin, cab, PEPCase and S-E9 small subunit RuBP carboxylase promoter. In still further embodiments of the invention, the expression vector may comprise any selectable marker, for example, a selectable marker selected from the group consisting of phosphinothricin acetyltransferase, glyphosate resistant EPSPS, aminoglycoside phosphotransferase, hygromycin phosphotransferase, neomycin phosphotransferase, dalapon dehalogenase, bromoxynil resistant nitrilase, anthranilate synthase and glyphosate oxidoreductase.
The expression vector may be either circular, for example, as in the case of a plasmid vector, or could be a linear nucleic acid segment, such as an expression cassette isolated from a plasmid. In particular embodiments of the invention, the vector is a plasmid vector. The expression vector may further comprise other elements, such as a nucleic acid sequence encoding a transit peptide, or potentially any terminator, for example, a heterologous terminator such as the nos terminator.
In still yet another aspect, the invention provides a transgenic plant comprising a stably transformed expression vector, such as those described above. The transgenic plant may be any type of plant, and in particular embodiments of the invention is a tomato plant. In further embodiments of the invention, the transgenic plant may be a fertile R0 transgenic plant. Also included in the invention is a seed of such a fertile R0 transgenic plant, wherein said seed comprises said expression vector. The transgenic plant may be a progeny plant of any generation of a fertile R0 transgenic plant, wherein said R0 transgenic plant comprises said expression vector. The invention also includes a seed of such a progeny plant, wherein said seed comprises said expression vector.
In still yet another aspect, the invention provides a crossed fertile transgenic plant prepared according to the method comprising the steps of: (i) obtaining a fertile transgenic plant comprising a selected DNA comprising a NOR gene; (ii) crossing said fertile transgenic plant with itself or with a second plant lacking said selected DNA to prepare the seed of a crossed fertile transgenic plant, wherein said seed comprises said selected DNA; and (iii) planting said seed to obtain a crossed fertile transgenic plant. In one embodiment of the invention, a seed is provided of such a crossed fertile transgenic plant, wherein said seed comprises said selected DNA. The crossed fertile transgenic plant may be of any species, for example, a tomato plant. The plant may also be inbred or hybrid.
In still yet another aspect, the invention provides a method of manipulating the phenotype of a plant comprising the steps of: (i) obtaining an expression vector comprising a NOR gene in sense or antisense orientation; (ii) transforming a recipient plant cell with said expression vector-and (iii) regenerating a transgenic plant from said recipient plant cell, wherein the phenotype of said plant is altered based on the expression of said NOR gene in sense or antisense orientation. Any method of transforming a plant may be used in accordance with the invention, including, microprojectile bombardment, PEG mediated transformation of protoplasts, electroporation, silicon carbide fiber mediated transformation, or Agrobacterium-mediated transformation. In particular embodiments of the invention, Agrobacterium-mediated transformation is used and the plant is a tomato plant.
In still yet another aspect, the invention provides a method of plant breeding comprising the steps of: (i) obtaining a transgenic plant comprising a selected DNA comprising a NOR gene, and (ii) crossing said transgenic plant with itself or a second plant. The plant may be of any species and may be inbred or hybrid. In particular embodiments of the invention, this method further comprises the steps of: (iii) collecting seeds resulting from said crossing; (iv) growing said seeds to produce progeny plants; (v) identifying a progeny plant comprising said selected DNA; and (vi) crossing said progeny plant with itself or a third plant. In one embodiment of the invention, the second plant and third plant are of the same genotype. The second and third plants may also be inbred plants.
In still yet another aspect, the invention provides a transgenic plant cell stably transformed with a selected DNA comprising a NOR gene. The cell may be from any plant species, for example, a cell from a tomato plant. The selected may comprise any of the NOR gene comprising nucleic acid compositions disclosed herein, for example, the expression vector compositions described herein above. Such compositions include the open reading frame of the NOR gene, as provided in SEQ ID NO:6 or SEQ ID NO:7 and demarcated in FIG. 6.